Tobacco addiction is a multifaceted biobehavioral phenomenon that is supported by the primary reinforcing effects of nicotine as well as by nicotine's ability to relieve anxiety. Our work and others have shown that activation of ?2 containing nicotinic acetylcholine receptors (?2*nAChRs;*denotes assembly with other subunits) promotes the primary reinforcing effects of nicotine, but less is known regarding how nicotine promotes anxiolysis in smokers. This proposal will test the hypothesis that inactivation of subsets of ?2*nAChRs by nicotine supports anxiolytic-like behavior. After activation, nAChRs become desensitized, and subactivating doses of nicotine preferentially desensitize nAChRs. This hypothesis is further supported by studies showing that low doses of nicotine lead to anxiolytic-like behavior whereas high doses promote anxiogenisis. A primary goal of these studies is to identify which subunits in combination with ?2 regulate anxiety-like behavior. ?2*nAChRs can be broken down by ?-conotoxin MII (?-CMII) sensitivity. The ?-CMII- sensitive ?6?3?2*nAChRs are selectively expressed in catecholaminergic nuclei with preferential expression on terminals in brain areas that regulate reward-like behavior. The ?-CMII insensitive ?4?2*nAChRs are more ubiquitously expressed in regions that also regulate anxiety-like behavior, including the amygdala and the lateral septum. We will use mice genetically altered to have a loss or gain of function of their ?4 and ?6 nAChRs to test if ?4?2*nAChRs preferentially regulate affective behaviors. These studies will further determine if nicotine acts through ?4?2*nAChRs or ?6?3?2*nAChRs to regulate lateral septal changes in intracellular signaling pathways, such as extracellular regulated kinase (ERK), that are implicated in regulation of stress. Using neurochemical and molecular manipulation of ERK signaling in the lateral septum, these studies will make a functional link between changes in ERK signaling and expression of anxiety-like behavior. Overlaid with our genetic technologies, these studies will identify if ?2*nAChR regulation of ERK is a critical mechanism by which ?2*nAChRs regulate anxiolysis or anxiogenisis. The proposed studies will substantially increase our understanding of which nicotinic subunits in combination with ?2 regulate anxiety behavior and determine whether these nAChRs exert their effects in the lateral septum. Collectively, this proposed work will provide insights into whether activation or inhibition of these receptors represents potential strategies for development of novel therapies to promote smoking cessation and to relieve anxiety. PUBLIC HEALTH RELEVANCE: Nicotinic receptors function largely as neuromodulators in the brain with consequent effects of their activation or inhibition on cognition, reward and affective behavior. Although most neurotransmitters exert their effects via second messenger systems, few studies have made functional links between nicotine-associated changes in intracellular signaling and behaviors that are relevant to nicotine and tobacco addiction. The proposed studies will identify selective nicotinic subunit regulation of signaling pathways that support affective behaviors.